Multimeric dual-modality breast cancer diagnostic agents

ABSTRACT

The present invention describes dual-modality probes. In particular, the present invention discloses hydroxyapatite specific multimeric bisphosphonate dual-modality MRI and optical probes.

FIELD OF THE INVENTION

The present invention discloses hydroxyapatite specific multimericbisphosphonate dual-modality MRI and optical probes.

BACKGROUND

Tissue calcification is an important biomarker for human disease, withmicrocalcifications being of paramount importance for the detection ofbreast cancer. Microcalcifications are of two major types. Type Icrystals, found more frequently in benign ductal cysts, are birefringentand colorless, and are composed of calcium oxalate {Morgan, 2005}. TypeII crystals, most often seen in proliferative lesions and associatedwith breast cancer cells, are composed of calcium hydroxyapatite, andare non-birefringent and basophilic {Haka, 2002}.

In the general population, breast cancer screening employs x-raymammography {Van Ongeval, 2006}. In 30% to 50% of cases,microcalcification is the hallmark for the presence of cancer {Morgan,2005}, although x-ray mammography cannot distinguish the chemical formof the calcium salts present, and therefore relies on the pattern ofcrystal deposition {Stomper, 2003}.

Mammography is currently the gold standard for the early detection ofbreast cancer {Bassett, 1992; Bassett 2000}. However, mammographysuffers from relatively low sensitivity and specificity {Mavroforou,2006}, and mammographic screening is limited in certain patientpopulations {Huo, 2002} and breast densities {Kolb, 2002}. Theselimitations have spurred interest in alternate modalities to detectbreast cancer.

SUMMARY

Imaging methods such as magnetic resonance and optical that couldnoninvasively and repeatedly measure integrin expression, would beuseful in characterizing tumors and in monitoring responses totherapeutic agents. In general, optical imaging methods have highsensitivity and are cost effective at the cell/tissue level. However,most optical imaging apparatus lacks the capacity of tomographic imagereconstruction, and therefore 3-dimensional localization of signals inintact tissues/organs has rarely been achieved noninvasively {Li, 2004}.Magnetic resonance imaging (MRI) offers the advantages of beingnoninvasive, tomographic, and high resolution. However, MRI contrastdependent on endogenous differences in water content and on relaxationtime in the tissue of interest. The specificity and sensitivity of MRIis enhanced by contrast agents based on paramagnetic metals such asgadolinium {Bottrill, 2006}.

The co-registration of different molecular imaging modalities providescomplementary information. Thus development of multifunctional probesfor concurrent imaging applications has become an attractive area.Combining the excellent 3D spatial resolution and unlimited depthpenetration of MRI with very high sensitivity of near infrared (NIR)optical imaging should serve to traverse shortcomings of each technology{Massoud, 2003}. NIR tomography has shown the ability to localizechanges in functional tissue parameters in vivo, and MRI has theadvantage of offering anatomical information about the layered adiposeand glandular tissue structure of the breast.

Magnetic resonance and NIR optical concurrent imaging of breast cancermicrocalcification has been elusive. Bisphosphonates bind avidly tohydroxyapatite bone mineral surfaces {van Beek, 1998} and have bothdiagnostic {Ogawa, 2005; Lam, 2007} and therapeutic uses {Lipton, 2000}.Bisphosphonates are analogues of endogenous pyrophosphates in which thehydrolysable oxygen atom that separates the two phosphate groups isreplaced with a more stable carbon atom. The P—C—P structure isresponsible for giving bisphosphonates their high affinity for bone,which can be further enhanced by addition of a hydroxyl group at thecentral carbon atom {van Beek, 1998}. An ideal molecular imagingtargeting ligand for magnetic resonance and NIR optical concurrentdetection of hydroxyapatite microcalcification of breast cancer is1-hydroxy-1,1-bisphosphonate derivatives {Bhushan, 2008; Bhushan, 2007},which shows remarkable specificity for hydroxyapatite, which is commonin malignant breast disease, over other calcium salts, such as calciumoxalate, which is typically deposited in benign lesions and is rarelyseen in malignancies {Morgan, 2005; Baker, 2007}.

Nature often takes advantage of multimerization to decrease ligandoff-rate and improve affinity of cell surface binders {Kitov, 2003;Mammen, 1998}. There is a general need to find suitable scaffolds forthe assembly of multiple targeting ligands and contrast agents in hopethat multimerization would improve the performance of cancer specificligands.

Several different multivalent scaffolds have been used successfully inpast particularly for applications in carbohydrate/lectin interactions{Lindhorst, 2002; Lundquist, 2002} but also for peptide/proteininteractions {Wright, 2001} and in context of tumor targeting {Carlson,2007; Thumshirn, 2003}. Among these scaffolds are small molecules withfew conjugation sites (˜2-10) and larger systems like dendrimers{Voegtle, 2007} and polymers {Haag, 2006}.

The present invention describes a development of multimerichydroxyapatite specific dual-modality MRI and optical probes. A systemto combine high sensitivity of NIR optical imaging in a planartomographic geometry with 3D spatial resolution and unlimited depthpenetration of MRI for breast imaging is described. In particular, thepresent invention describes a chemical system for the efficientproduction of a tri-functional agent comprised of a NIR fluorophore foroptical imaging, a metal chelate for simultaneous MRI, and abisphosphonate specific for hydroxyapatite, the major calcium saltproduced during osteoblastosis. The multimeric hydroxyapatite specificdual-modality MRI and optical probes could theoretically bindmultivalently and thus more avidly to target hydroxyapatite.Multifunctional probes for concurrent imaging applications couldtraverse shortcomings of each technology and could provide complementaryinformation.

In one aspect of present invention, an organic chelating ligand isreacted with a trifunctional linker moiety, having primary amine andcarboxylic acid functional groups, followed by conjugation with abisphosphonate to result in a bisphosphonate conjugated organicchelating ligand. Deprotection of one or more functional groups on abisphosphonate conjugated organic chelating ligand yields one or morefree functional groups. Chelation of a metal ion on one or more freefunctional groups results in a metal chelate. Conjugation of a NIRfluorophore on a metal chelate results in a dual-modality MRI andoptical probe (FIG. 1). In such aspect, trifunctional linker moiety 2 isamino acid, polymer, or dendrimer. L₁, L₂, and L₃ are independentlyselected from alkane, polyethylene glycol, and polypropylene glycol.Metal ion, M is independently selected from Cu, Fe, In, Tm, Yb, Y, Gd,Eu, and a lanthanide. R is t-butyl ester, ester, or hydrogen. R¹ is Boc,Fmoc, Ac, Cbz, Bz, and Bn. In one embodiment, amino acid is naturalamino acid. In some embodiment, amino acid is unnatural amino acid. Insome embodiments, alkane is C0-C20 straight chain carbon unit. In someembodiments, polyethylene glycol is 1 to 20 ethylene glycol unit. Insome embodiments, polypropylene glycol is 1 to 20 propylene glycol unit.In some embodiments, bisphosphonate, BP is independently selected fromalendronate, etidronate, ibandronate, incadronate, neridronate,olpadronate, pamidronate, risedronate, tiludronate, and zoledronate. Insome embodiments, IRDye is a NIR fluorophore independently selected fromthe group of IRDye 78, IRDye 700DX, VivoTag-S 750, VivoTag 800,VivoTag-S 680, DY-750, DY-682, DY-675, Cypate, Cy7, Alexa Fluor 750, andAlexa Fluor 680.

In an another aspect of present invention, a bisphosphonate isconjugated with multivalent scaffold followed by deprotection of aminoprotecting group to generate an amine containing bisphosphonateconjugated multivalent scaffold. Reaction of an amine containingbisphosphonate conjugated multivalent scaffold with a trifunctionallinker moiety conjugated organic chelating ligand 3 (FIG. 1) results inan organic chelating ligand containing bisphosphonate conjugatedmultivalent scaffold 9 (FIG. 2). Deprotection of one or more functionalgroups on an organic chelating ligand containing bisphosphonateconjugated multivalent scaffold, followed by a metal chelation andconjugation with a NIR fluorophore yields a multimeric dual-modality MRIand optical probe. In such aspect, R¹ is independently selected fromBoc, Fmoc, Ac, Cbz, Bz, and Bn. R is t-butyl ester, ester, or hydrogen.R², R³, and R⁴ are bisphosphonates or OH. Metal ion, M is independentlyselected from Cu, Fe, In, Tm, Yb, Y, Gd, Eu, and a lanthanide. L₁, L₂,L₃, L₄, and L₅ are linkers independently selected from alkane, aminoacid, —NHCO(CH₂)₅—, polyethylene glycol, and polypropylene glycol. Inone embodiment, amino acid is natural amino acid. In some embodiments,amino acid is unnatural amino acid. In some embodiments, an alkane isC0-C20 straight chain carbon unit. In some embodiments, polyethyleneglycol is 1 to 20 ethylene glycol unit. In some embodiments,polypropylene glycol is 1 to 20 propylene glycol unit. In someembodiments, bisphosphonate is independently selected from alendronate,etidronate, ibandronate, incadronate, neridronate, olpadronate,pamidronate, risedronate, tiludronate, and zoledronate. In someembodiments, IRDye is a NIR fluorophore independently selected from thegroup of IRDye 78, IRDye 700DX, VivoTag-S 750, VivoTag 800, VivoTag-S680, DY-750, DY-682, DY-675, Cypate, Cy7, Alexa Fluor 750, and AlexaFluor 680.

The present invention describes dual-modality probes. Particularly, thepresent invention discloses hydroxyapatite specific multimericbisphosphonate dual-modality MRI and optical probes. Dual-modalityprobes of present invention provide complementary information. The majormedical application of present invention is in the high sensitivitysimultaneous NIR optical and MRI detection of tissue calcification,especially microcalcification in breast cancer, without the need forionizing radiation.

BRIEF DESCRIPTION OF DRAWING

FIG. 1 represents a dual-modality probe in which a NIR fluorophore isconjugated after a metal ion chelation.

FIG. 2 represents a multimeric dual-modality probe in which a NIRfluorophore is conjugated after a metal ion chelation.

FIG. 3 represents a dual-modality probe in which a NIR fluorophore isconjugated before a reaction with a metal chelate.

FIG. 4 represents a multimeric dual-modality probe in which a NIRfluorophore is conjugated before a reaction with a metal chelate.

FIG. 5 represents a synthetic scheme for preparation of[Gd-DOTA]-Lys-IRDye-Alen monomer.

FIG. 6 represents a synthetic scheme for preparation ofAsp-IRDye-Ad-Alen trimer.

FIG. 7 represents a synthetic scheme for preparation of[Gd-DOTA]-Asp-IRDye-Ad-Alen trimer.

DETAILED DESCRIPTION

In a present invention, synthetic strategy is developed for multimericbisphosphonate dual-modality probes for targeted imaging of breastcancer microcalcification. Particularly, present invention describes achemical system for the efficient production of a tri-functional agentcomprised of a NIR fluorophore for optical imaging, a metal chelate forsimultaneous MRI, and a bisphosphonate specific for hydroxyapatite, themajor calcium salt produced during osteoblastosis. Dual-modality probesof present invention allows cross validation and direct comparisonbetween MRI and NIR optical imaging.

The multimeric bisphosphonate dual-modality probes of present inventionare prepared according to the methods known in the art, as illustratedin FIGS. 1-4 and described for specific compounds in examples 1-3.Products are characterized by analytical HPLC, NMR, and LC-MS. Monomericdual-modality probes are obtained in typical yields of 55-65% andtrimeric dual-modality probes are obtained in typical yields of 20-30%.

FIG. 1 of present invention describes a synthetic scheme for adual-modality probe in which a NIR fluorophore is conjugated after ametal ion chelation. An organic chelating ligand is reacted with atrifunctional linker moiety, followed by conjugation with abisphosphonate to result in a bisphosphonate conjugated organicchelating ligand. Deprotection of one or more functional groups on abisphosphonate conjugated organic chelating ligand yields one or morefree functional groups. Chelation of a metal ion on one or more freefunctional groups results in a metal chelate, followed by conjugation ofa NIR fluorophore to result in a dual-modality MRI and optical probe.

FIG. 2 of present invention describes a synthetic scheme for amultimeric dual-modality probe in which a NIR fluorophore is conjugatedafter a metal ion chelation. A bisphosphonate is conjugated with amultivalent scaffold, followed by deprotection of an amino protectinggroup to generate an amine containing bisphosphonate conjugatedmultivalent scaffold. Reaction of an amine containing bisphosphonateconjugated multivalent scaffold with a trifunctional linker moietyconjugated organic chelating ligand 3 (FIG. 1) results in an organicchelating ligand containing bisphosphonate conjugated multivalentscaffold 9. Deprotection of one or more functional groups on an organicchelating ligand containing bisphosphonate conjugated multivalentscaffold, followed by a metal chelation and conjugation with a NIRfluorophore yields a multimeric dual-modality MRI and optical probe.

FIG. 3 of present invention describes a synthetic scheme for adual-modality probe in which a NIR fluorophore is conjugated before areaction with a metal chelate. A bisphosphonate is conjugated with atrifunctional linker moiety, followed by deprotection and conjugationwith a NIR fluorophore to result in a NIR fluorophore containingbisphosphonate conjugated carboxylic acid precursor 14. Reaction of ametal chelate 16 with a NIR fluorophore containing bisphosphonateconjugated carboxylic acid precursor 14 results in a dual-modality probe17.

FIG. 4 of present invention describes a synthetic scheme for amultimeric dual-modality probe in which a NIR fluorophore is conjugatedbefore a reaction with a metal chelate. A bisphosphonate is conjugatedwith a multivalent scaffold followed by deprotection of an aminoprotecting group to generate an amine containing bisphosphonateconjugated multivalent scaffold. Reaction of an amine containingbisphosphonate conjugated multivalent scaffold with a trifunctionallinker moiety, followed by deprotection and NIR fluorophore conjugationresults in a NIR fluorophore containing bisphosphonate conjugatedmultivalent scaffold 20. Reaction of a metal chelate 16 with a NIRfluorophore containing bisphosphonate conjugated multivalent scaffold 20results in a multimeric dual-modality probe 21.

In one aspect, the present invention provides a dual-modality probehaving a formula selected from the group of:

In such aspect,

BP is a bisphosphonate,

L₁, L₂, L₃, L₄, and L₅ are linkers,

IRDye is a near infrared dye with wavelength in the range of 700-900 nm,and

is a metal chelate independently selected from:

In one embodiment, linkers are independently selected from alkane, aminoacid, —NHCO(CH₂)₅—, polyethylene glycol, and polypropylene glycol. Insome embodiments, amino acid is natural amino acid. In some embodiments,amino acid is unnatural amino acid. In some embodiments, alkane isC0-C20 straight chain carbon unit. In some embodiments, polyethyleneglycol is 1 to 20 ethylene glycol unit. In some embodiments,polypropylene glycol is 1 to 20 propylene glycol unit. In someembodiments, M is independently selected from Cu, Fe, In, Tm, Yb, Y, Gd,Eu, and a lanthanide. In some embodiments, near infrared dye isindependently selected from the group of IRDye 78, IRDye 700DX,VivoTag-S 750, VivoTag 800, VivoTag-S 680, DY-750, DY-682, DY-675,Cypate, Cy7, Alexa Fluor 750, and Alexa Fluor 680. In some embodiments,bisphosphonate is independently selected from the group of alendronate,etidronate, ibandronate, incadronate, neridronate, olpadronate,phosphonate, pamidronate, risedronate, tiludronate, and zoledronate.

In an another aspect, the present invention provides a method of makinga dual-modality probe. The method involves steps of:

(a) Starting synthesis with an organic chelating ligand selected fromthe group of:

where, R is t-butyl ester, ester, or hydrogen,and

R¹ is

(b) reacting a organic chelating ligand with a trifunctional linkermoiety to result in a trifunctional linker moiety conjugated organicchelating ligand, (c) reacting a trifunctional linker moiety conjugatedorganic chelating ligand with a bisphosphonate to result in abisphosphonate conjugated organic chelating ligand, (d) deprotecting oneor more functional groups on a bisphosphonate conjugated organicchelating ligand to yield one or more free functional groups, (e)chelating a metal ion on one or more free functional groups to result ina metal chelate, and (f) conjugating a NIR fluorophore with a metalchelate to result in a dual-modality probe.

In one embodiment, bisphosphonate is selected from the group of:

where, L₁, and L₂ are independently selected from alkane, amino acid,—NHCO(CH₂)₅—, polyethylene glycol, and polypropylene glycol, and R², R³,and R⁴ are independently selected from alendronate, etidronate,ibandronate, incadronate, neridronate, olpadronate, pamidronate,risedronate, tiludronate, zoledronate, and OH. In some embodiments,trifunctional linker moiety is selected from amino acid, polymer, anddendrimer. In some embodiments, amino acid is natural amino acid. Insome embodiments, amino acid is unnatural amino acid. In someembodiments, alkane is C0-C20 straight chain carbon unit. In someembodiments, polyethylene glycol is 1 to 20 ethylene glycol unit. Insome embodiments, polypropylene glycol is 1 to 20 propylene glycol unit.In some embodiments, metal ion is independently selected from Cu, Fe,In, Tm, Yb, Y, Gd, Eu, and a lanthanide. In some embodiments, NIRfluorophore is independently selected from the group of IRDye 78, IRDye800CW, IRDye 700DX, VivoTag-S 750, VivoTag 800, VivoTag-S 680, DY-750,DY-682, DY-675, Cypate, Cy7, Alexa Fluor 750, and Alexa Fluor 680. Insome embodiments, bisphosphonate is independently selected from thegroup of alendronate, etidronate, ibandronate, incadronate, neridronate,olpadronate, phosphonate, pamidronate, risedronate, tiludronate, andzoledronate.

In an another aspect, the present invention provides a method of makinga dual-modality probe. The method involves steps of:

(a) Starting a synthesis with reacting a bisphosphonate with atrifunctional linker moiety to result in a trifunctional linker moietyconjugated bisphosphonate, (b) deprotecting one or more functionalgroups on a trifunctional linker moiety conjugated bisphosphonate toyield one or more free functional groups, (c) conjugating a NIRfluorophore to result in a NIR fluorophore containing trifunctionallinker moiety conjugated bisphosphonate carboxylic acid precursor, (d)providing a metal chelate selected from the group of:

and(e) reacting a metal chelate with a NIR fluorophore containingtrifunctional linker moiety conjugated bisphosphonate carboxylic acidprecursor under a condition capable of forming an amide bond to resultin a dual-modality probe.

In one embodiment, bisphosphonate is selected from the group of:

where, L₁, and L₂ are independently selected from alkane, amino acid,—NHCO(CH₂)₅—, polyethylene glycol, and polypropylene glycol, and R², R³,and R⁴ are independently selected from alendronate, etidronate,ibandronate, incadronate, neridronate, olpadronate, pamidronate,risedronate, tiludronate, zoledronate, and OH. In some embodiments,trifunctional linker moiety is selected from amino acid, polymer, anddendrimer. In some embodiments, amino acid is natural amino acid. Insome embodiments, amino acid is unnatural amino acid. In someembodiments, alkane is C0-C20 straight chain carbon unit. In someembodiments, polyethylene glycol is 1 to 20 ethylene glycol unit. Insome embodiments, polypropylene glycol is 1 to 20 propylene glycol unit.In some embodiments, M is independently selected from Cu, Fe, In, Tm,Yb, Y, Gd, Eu, and a lanthanide. In some embodiments, NIR fluorophore isindependently selected from the group of IRDye 78, IRDye 700DX,VivoTag-S 750, VivoTag 800, VivoTag-S 680, DY-750, DY-682, DY-675,Cypate, Cy7, Alexa Fluor 750, and Alexa Fluor 680. In some embodiments,bisphosphonate is independently selected from the group of alendronate,etidronate, ibandronate, incadronate, neridronate, olpadronate,phosphonate, pamidronate, risedronate, tiludronate, and zoledronate.

With high quantum yields, the spectral properties of dual-modality MRIand optical probes demonstrates peak absorptions (770-780 nm) andemission (790-800 nm), located within the “NIR window,” an area of theelectromagnetic spectrum that maximizes photon penetration and recoveryin living tissue.

The multimeric bisphosphonate dual-modality probes generated by presentinvention can be used for, e.g., optical, magnetic resonance,radioimmuno, PET, and SPECT applications for detection, imaging andtreatment of breast cancer microcalcification and other abnormalities.In particular, multimeric bisphosphonate dual-modality MRI and opticalprobes generated by present invention are hydroxyapatite specific fordetection of breast cancer microcalcification. Specifically,trimerization of bisphosphonate using a multivalent scaffold results insignificantly higher in vitro specificity for hydroxyapatite, a majormineral component of calcification and normal bone, over other calciumsalts, in comparison to monomeric bisphosphonate dual-modality probewithout a multivalent scaffold.

To determine the selectivity and specificity of [Gd-DOTA]-Lys-IRDye-Alenmonomer and [Gd-DOTA]-Asp-IRDye-Ad-Alen trimer for hydroxyapatite, amajor mineral component of calcifications and normal bone, over othercalcium salts, in the present invention an incubation of equal quantityeach of Ca-hydroxyapatite, Ca-pyrophosphate, Ca-phosphate, Ca-oxalate,and Ca-carbonate salts with [Gd-DOTA]-Lys-IRDye-Alenmonomer/[Gd-DOTA]-Asp-IRDye-Ad-Alen trimer in phosphate buffered saline(PBS) is performed. NIR fluorescence images are acquired afterincubation and washing of crystals, dual-modality probes has many foldhigher specificity for hydroxyapatite over other calcium salts found inthe body and permits high sensitivity detection of hydroxyapatite.

EXAMPLES 1. Preparation of [Gd-DOTA]-Lys-IRDye-Alen Monomer FIG. 5DOTA(OtBu)₃-Boc-Lys:

To a solution of Boc-L-Lysine (0.06 mmol) in 0.4 mL DMF at 0° C., isadded triethylamine (0.12 mmol) followed by dropwise addition ofDOTA(OtBu)₃ NHS ester (0.05 mmol) in 0.5 mL DMF for 10 min withstirring. After 10 min, the ice bath is removed and stirring iscontinued at room temperature (RT) for 6 h. The reaction mixture ispoured over 2 mL ice-cold water and purified by preparative HPLC toobtain DOTA(OtBu)₃-Boc-Lys 22.

DOTA(tBu)₃-BOC-Lys-Alen:

Alendronic acid (0.05 mmol), HCTU (0.05 mmol), and N-methylmorpholine(NMM; 0.20 mmol) are added at RT under N₂ atmosphere to 0.05 mmolDOTA(OtBu)₃-Boc-Lys 22 in DMSO (1 mL). After stirring for 3 h at RT, thereaction mixture is poured over 3 mL ice-cold water and an intermediateDOTA(tBu)₃-Boc-Lys-Alen 23 is purified by preparative HPLC.

DOTA-Lys-Alen:

DOTA(tBu)₃-Boc-Lys-Alen 23 (0.045 mmol) is taken in 95% trifluoroaceticacid (TFA, 1 mL). The solution is stirred at RT for 6 h then removed theacid by N₂ stream. After lyophilization, DOTA-Lys-Alen 24 is obtainedwithout further purification as a white powder.

[Gd-DOTA]-Lys-Alen:

The chelation of Gd³⁺ is performed by adding 50 μL of 1 M GdCl₃ in waterto a solution of DOTA-Lys-Alen 24 (0.04 mmol) in 950 μL of 0.5 M HAc/Ac⁻buffer, pH 5.5. The reaction mixture is stirred at RT for 12 h. Thecompound is purified by preparative HPLC to obtain [Gd-DOTA]-Lys-Alen25.

[Gd-DOTA]-Lys-IRDye-Alen Monomer:

To [Gd-DOTA]-Lys-Alen 25 (0.01 mmol) in 1 mL DMSO, is added NHS ester ofthe NIR fluorophore IRDye 78 (IRDye-NHS, 0.01 mmol) andN,N-diisopropylethylamine (0.05 mmol) at 0° C. under nitrogenatmosphere. The stirring is continued for 2 h at RT in the dark. Thereaction mixture is poured over 4 mL ice-cold water, purified by HPLCand concentrated on an Oasis HLB desalting cartridge as describedpreviously {Bhushan, 2008}. On lyophilization a bright green solidreaction component, [Gd-DOTA]-Lys-IRDye-Alen monomer 26 is obtained.

2. Preparation of Asp-IRDye-Ad-Alen Trimer FIG. 6 Ad-Alen Trimer:

Alendronic acid (0.06 mmol) is dissolved in 1 mL of DMSO andtriethylamine (0.30 mmol). After 5 min, a solution of Boc-NH-Ad-Tri-NHS(0.015 mmol) {Humblet, 2009} in 0.2 mL of DMSO is added. The reactionmixture is stirred at RT for 16 h. The compound 27 is purified afterdilution into a final volume of 5 mL with ice-cold water by preparativeHPLC system. After lyophilization, product 27 is treated with 95% TFA (1mL) for 3 h. Excess TFA is removed under nitrogen and afterlyophilization the Ad-Alen trimer 28 is obtained.

Asp-Ad-Alen Trimer:

To Ad-Alen trimer 28 (0.01 mmol) in 0.5 mL DMF at 0° C., is addedtriethylamine (0.15 mmol) followed by dropwise addition ofBoc-L-Asp(OtBu)-OSu (0.01 mmol) in 0.4 mL DMF for 10 min with stirring.After 10 min, the ice bath is removed and stirring is continued at RTfor 6 h. The reaction mixture is poured over 2 mL ice-cold water andpurified by preparative HPLC to obtain compound 29. Afterlyophilization, compound 29 is treated with 95% TFA (1 mL) for 6 h.Excess TFA is removed under nitrogen and after lyophilization theAsp-Ad-Alen trimer 30 is obtained.

Asp-IRDye-Ad-Alen Trimer:

To Asp-Ad-Alen trimer 30 (0.01 mmol) in 1 mL DMSO, is added NHS ester ofthe NIR fluorophore IRDye 78 (IRDye-NHS, 0.01 mmol) andN,N-diisopropylethylamine (0.15 mmol) at 0° C. under nitrogenatmosphere. The stirring is continued for 2 h at RT in the dark. Thereaction mixture is poured over 4 mL ice-cold water, purified by HPLCand concentrated on an Oasis HLB desalting cartridge as describedpreviously {Bhushan, 2008}. On lyophilization a bright green solidreaction component, Asp-IRDye-Ad-Alen trimer 31 is obtained.

3. Preparation of [Gd-DOTA]-Asp-IRDye-Ad-Alen Trimer FIG. 7DOTA(OtBu)₃-Boc-Diaminoethane:

To a solution of Boc-1,2-diaminoethane (0.06 mmol) in 0.4 mL DMF at 0°C., is added triethylamine (0.12 mmol) followed by dropwise addition ofDOTA(OtBu)₃ NHS ester (0.05 mmol) in 0.5 mL DMF for 10 min withstirring. After 10 min, the ice bath is removed and stirring iscontinued at RT for 6 h. The reaction mixture is poured over 2 mLice-cold water and purified by preparative HPLC to obtainDOTA(OtBu)₃-Boc-diaminoethane 32.

DOTA-Diaminoethane:

DOTA(OtBu)₃-Boc-diaminoethane 32 (0.045 mmol) is taken in 95% TFA (1mL). The solution is stirred at RT for 6 h then removed the acid by N₂stream. After lyophilization, DOTA-diaminoethane 33 is obtained withoutfurther purification as a white powder.

[Gd-DOTA]-Diaminoethane:

The chelation of Gd³⁺ is performed by adding 50 μL of 1 M GdCl₃ in waterto a solution of DOTA-diaminoethane 33 (0.04 mmol) in 950 μL of 0.5 MHAc/Ac⁻ buffer, pH 5.5. The reaction mixture is stirred at RT for 12 h.The compound is purified by preparative HPLC to obtain[Gd-DOTA]-diaminoethane 34.

[Gd-DOTA]-Asp-IRDye-Ad-Alen Trimer:

To a solution of Asp-IRDye-Ad-Alen trimer 31 (0.01 mmol) in 1 mL DMSO at0° C., is added HCTU (0.01 mmol) and NMM (0.15 mmol) followed bydropwise addition of [Gd-DOTA]-diaminoethane 34 (0.01 mmol) in 0.5 mLDMF for 10 min with stirring. After 10 min, the ice bath is removed andstirring is continued at RT for 2 h in the dark. The reaction mixture ispoured over 2 mL ice-cold water and purified by preparative HPLC toobtain [Gd-DOTA]-Asp-IRDye-Ad-Alen trimer 35.

4. In Vitro Calcium Salt Specificity Experiments

5 mg/mL of hydroxyapatite or the phosphate, oxalate, carbonate, andpyrophosphate salts of calcium are separately incubated with 100 nM[Gd-DOTA]-Asp-IRDye-Ad-Alen trimer in 100 mL PBS for 30 min withcontinuous vortexing at RT. Crystals are washed 4 times with a 100-foldexcess of PBS, centrifuged and visualized using the NIR fluorescenceimaging system {Bhushan, 2008) at a fluence rate of 5 mW/cm². All NIRfluorescence images have identical exposure times and normalizations.Similarly, calcium salt binding study is performed withGd-DOTA]-Lys-IRDye-Alen monomer in a same condition.

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What is claimed is:
 1. A dual-modality probe having a formula selectedfrom the group consisting of:

wherein

BP is a bisphosphonate; L₁, L₂, L₃, L₄, and L₅ are linkers; IRDye is anear infrared dye with wavelength in the range of 700-900 nm; and

is a metal chelate independently selected from:


2. The dual-modality probe of claim 1, wherein said linkers are selectedfrom the group consisting of alkane, amino acid, —NHCO(CH₂)₅—,polyethylene glycol, and polypropylene glycol.
 3. The dual-modalityprobe of claim 1, wherein said dual-modality probe is in a form ofpharmaceutically acceptable salts, hydrates, and solvates.
 4. Thedual-modality probe of claim 1, wherein M is selected from the groupconsisting of Cu, Fe, In, Mn, Tm, Yb, Y, Gd, Eu, and a lanthanide. 5.The dual-modality probe of claim 1, wherein said near infrared dye isselected from the group consisting of IRDye 78, IRDye 800CW, IRDye700DX, VivoTag-S 750, VivoTag 800, VivoTag-S 680, DY-750, DY-682,DY-675, Cypate, Cy7, Alexa Fluor 750, and Alexa Fluor
 680. 6. Thedual-modality probe of claim 1, wherein said bisphosphonate is selectedfrom the group consisting of alendronate, etidronate, ibandronate,incadronate, neridronate, olpadronate, phosphonate, pamidronate,risedronate, tiludronate, and zoledronate.
 7. A method of making adual-modality probe, said method comprising: (a) providing an organicchelating ligand, wherein said organic chelating ligand selected fromthe group of:

wherein R is t-butyl ester, ester, or hydrogen; and R¹ is

(b) reacting said organic chelating ligand with a trifunctional linkermoiety to result in a trifunctional linker moiety conjugated organicchelating ligand; (c) reacting said trifunctional linker moietyconjugated organic chelating ligand with a bisphosphonate to result in abisphosphonate conjugated organic chelating ligand; (d) deprotecting oneor more functional groups on said bisphosphonate conjugated organicchelating ligand to yield one or more free functional groups; (e)chelating a metal ion on said one or more free functional groups toresult in a metal chelate; and (f) conjugating a near infraredfluorophore with said metal chelate to result in said dual-modalityprobe.
 8. The method of claim 7, wherein said trifunctional linkermoiety is amino acid, polymer, or dendrimer.
 9. The method of claim 7,wherein said near infrared fluorophore is selected from the groupconsisting of IRDye 78, IRDye 700DX, VivoTag-S 750, VivoTag 800,VivoTag-S 680, DY-750, DY-682, DY-675, Cypate, Cy7, Alexa Fluor 750, andAlexa Fluor
 680. 10. The method of claim 7, wherein said metal ion isselected from the group consisting of Cu, Fe, In, Mn, Tm, Yb, Y, Gd, Eu,and a lanthanide.
 11. The method of claim 7, wherein said metal ion andsaid near infrared fluorophore are conjugated for concurrent magneticresonance and near infrared optical imaging.
 12. The method of claim 7,wherein said bisphosphonate is selected from the group consisting ofalendronate, etidronate, ibandronate, incadronate, neridronate,olpadronate, phosphonate, pamidronate, risedronate, tiludronate, andzoledronate.
 13. The method of claim 7, wherein said bisphosphonate isselected from the group consisting of:

wherein L₁, and L₂ are independently selected from alkane, amino acid,—NHCO(CH₂)₅—, polyethylene glycol, and polypropylene glycol; and R², R³,and R⁴ are independently selected from alendronate, etidronate,ibandronate, incadronate, neridronate, olpadronate, pamidronate,risedronate, tiludronate, zoledronate, and OH.
 14. A method of making adual-modality probe, said method comprising: (a) reacting abisphosphonate with a trifunctional linker moiety to result in atrifunctional linker moiety conjugated bisphosphonate; (b) deprotectingone or more functional groups on said trifunctional linker moietyconjugated bisphosphonate to yield one or more free functional groups;(c) conjugating a near infrared fluorophore to result in a near infraredcontaining trifunctional linker moiety conjugated bisphosphonatecarboxylic acid precursor; (d) providing a metal chelate, wherein saidmetal chelate selected from the group of:

and (e) reacting said metal chelate with said near infrared containingtrifunctional linker moiety conjugated bisphosphonate carboxylic acidprecursor under a condition capable of forming an amide bond to resultin said dual-modality probe.
 15. The method of claim 14, wherein saidtrifunctional linker moiety is amino acid, polymer, or dendrimer. 16.The method of claim 14, wherein said near infrared fluorophore isselected from the group consisting of IRDye 78, IRDye 700DX, VivoTag-S750, VivoTag 800, VivoTag-S 680, DY-750, DY-682, DY-675, Cypate, Cy7,Alexa Fluor 750, and Alexa Fluor
 680. 17. The method of claim 14,wherein M is selected from the group consisting of Cu, Fe, In, Mn, Tm,Yb, Y, Gd, Eu, and a lanthanide.
 18. The method of claim 14, whereinsaid dual-modality probe is in a form of pharmaceutically acceptablesalts, hydrates, and solvates.
 19. The method of claim 14, wherein saidbisphosphonate is selected from the group consisting of alendronate,etidronate, ibandronate, incadronate, neridronate, olpadronate,phosphonate, pamidronate, risedronate, tiludronate, and zoledronate. 20.The method of claim 14, wherein said bisphosphonate is selected from thegroup consisting of:

wherein L₁, and L₂ are independently selected from alkane, amino acid,—NHCO(CH₂)₅—, polyethylene glycol, and polypropylene glycol; and R², R³,and R⁴ are independently selected from alendronate, etidronate,ibandronate, incadronate, neridronate, olpadronate, phosphonate,pamidronate, risedronate, tiludronate, zoledronate, and OH.